347 Bakin Karfe naɗaɗɗen nau'in sinadarai, Gano sabon interferon mai amsawa ɗan adam leukocyte antigen-A (HLA-A) sunadaran chaperone ta amfani da haɗin haɗin kai (CLMS)

Na gode da ziyartar Nature.com.Kuna amfani da sigar burauza tare da iyakancewar tallafin CSS.Don ƙwarewa mafi kyau, muna ba da shawarar ku yi amfani da sabuntar burauza (ko kuma musaki Yanayin dacewa a cikin Internet Explorer).Bugu da ƙari, don tabbatar da goyon baya mai gudana, muna nuna shafin ba tare da salo da JavaScript ba.
Sliders suna nuna labarai uku a kowane faifai.Yi amfani da maɓallan baya da na gaba don motsawa ta cikin nunin faifai, ko maɓallan masu sarrafa nunin faifai a ƙarshen don matsawa ta kowane faifan.

Bayanin Samfura

Bakin Karfe 347L Coil Tubes, Karfe Matsayi: SS347L

Tsarin siginar siginar interferon yana haifar da amsawar cytokine mai ƙarfi ga nau'ikan siginar ƙwayoyin cuta da ƙwayoyin cuta daga yanayin yanayi, wanda ke haifar da ƙaddamar da ƙananan ƙwayoyin ƙwayoyin cuta na interferon-inducible.Mun yi amfani da DSS-mai shiga tsakani na giciye mass spectrometry (CLMS) don gano sabbin hulɗar sunadaran sunadaran gina jiki a cikin yanki na sunadaran da ke haifar da interferon.Bugu da ƙari ga sunadaran da ake sa ran interferon-inducible sunadaran, mun kuma gano sabon abu na interferon-inducible adcts na canonical interferon-inducible sunadaran kamar MX1, USP18, OAS3, da STAT1.Mun mayar da hankali kan ingantaccen ingantaccen tsari na wani sabon saitin hanyoyin sadarwa na furotin mai iya haifar da interferon wanda furotin HLA-A (H2BFS-HLA-A-HMGA1) suka kirkira ta amfani da haɗin kai tare da ƙarin bincikensu ta amfani da ƙirar ƙirar ƙwayoyin cuta.Samfuran abubuwan da suka dace na hadaddun furotin sun bayyana wuraren hulɗa da yawa waɗanda ke nuna hulɗar da aka gano a cikin binciken CLMS.Tare, muna gabatar da binciken matukin jirgi na CLMS don gano sabbin rukunin sigina da interferon ya jawo, da kuma sa ido ga faɗuwar amfani da CLMS don gano sabbin hanyoyin hulɗar furotin a cikin ƙananan ƙwayoyin cuta.
Kafin a fara mayar da martanin rigakafi mai daidaitawa, tsarin tsaro na mahaifar mai masaukin baki yana ɗaukar martanin antimicrobial wanda dangin cytokines na alpha-helical da aka ɓoye da ake kira interferon (IFNs).Nau'in I IFN azuzuwan IFNa da IFNβ suna kunna martanin salula, gami da antiviral, proapoptotic, proinflammatory, da jihohin antiproliferative.A cikin mutane, 13 subtypes na IFNa an san su, duk sun taru akan chromosome 91. Abin mamaki, IFNα2 kawai an yi nazarin don amfani da asibiti.Kwanan nan, an biya kulawa ta musamman ga bincike akan wasu nau'ikan nau'ikan IFNα.Wani binciken da aka yi a baya-bayan nan ya nuna cewa IFNα14 yana daya daga cikin mafi tasiri isoforms a cikin ƙuntata HBV2 da HIV-13,4 kwafi idan aka kwatanta da canonical IFNα2 subtype.
An kafa cewa kunna nau'in I interferon receptor complexes (IFNAR1 da IFNAR2) suna haifar da watsawar sigina wanda Janus kinases TYK2 da JAK15,6 suka shiga tsakani.Waɗannan Janus kinases masu fassarar siginar phosphorylate da masu kunna furotin na rubutu (STAT1 da STAT2) akan ragowar tyrosine don fara SH2 yanki-mai daidaita heterodimerization6.Daga baya, IRF9 yana ɗaure STAT heterodimers don samar da hadaddun trimeric na IFN-stimulated factor 3 gene (ISGF3), wanda ke juyawa zuwa tsakiya kuma yana haifar da rubutun sama da 2000 interferon-stimulated genes (ISGs) 5,6,7,8.
ISGs sune kashin bayan tsarin rigakafi na halitta, musamman don mayar da martani ga harin hoto.A matsayin layin farko na kariya daga kamuwa da cutar hoto, ƙwayoyin sel suna hanzarta tura manyan mu'amala na sunadaran salula tare da fa'idodin ayyukan halitta.Waɗannan sunadaran sun haɗa da masu karɓar ƙirar ƙira, ƙwayoyin sigina, abubuwan rubutawa, da kuma sunadaran da ke da ayyukan rigakafin kamuwa da cuta kai tsaye, da kuma masu kula da martani mara kyau9.Yawancin bayanai game da ayyukan ISG sun fito ne daga allon aiki ta amfani da fuskar bangon waya 10,11 ko dabarun shiru na kwayoyin halitta (siRNA, RNAi da CRISPR) 12,13 wanda aka bayyana ko hana kowane ISGs kuma ana gwada ayyukansu akan ƙwayoyin cuta daban-daban.Ko da yake waɗannan binciken sun ƙayyade kaddarorin antiviral na ISG guda ɗaya, tushen tsarin kwayoyin halitta na kowane ISG ya kasance ba a san su ba.An yarda da cewa yawancin sunadaran suna yin hulɗa tare da cytokines ɗaya ko fiye don tabbatar da cikakken aiki, don haka ko dai ISGs suna hulɗa kai tsaye ko kuma hulɗar su suna yin sulhu ta hanyar sunadaran salula.Alal misali, binciken da aka yi kwanan nan na photocrosslinked proteomics ya gano ATPase VCP/p97 a matsayin babban abokin hulɗar IFITM3, wanda hanawa ya haifar da lahani a cikin rarrabawar lysosomal, juyawa, da jigilar kaya na IFITM3 tare da ƙwayoyin cuta 14.Yin amfani da immunoprecipitation, mun gano VAPA, furotin da ke hade da vesicle, a matsayin abokin hulɗa tare da IFITM1 / 2/3 wanda ke ƙaddamar da ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta, wannan ya tabbatar da wannan ta hanyar amfani da tsarin yisti biyu-hybrid.Taimakon Kimiyya 15, 16 .
Wani muhimmin tsari na nazarin halittu wanda ke da hannu a cikin kashe kamuwa da cuta da kuma mummunan canji shine gabatarwar antigen, wanda ke yin sulhu ta hanyar manyan kwayoyin halitta na histocompatibility (MHC).Peptides (8-12 amino acid tsawon) daga tsage-tsalle, da aka ƙare da wuri ko kuma ɓarna sunadaran sunadaran a cikin MHC-I heterodimer (wanda ya ƙunshi MHC-I nauyi da sarƙoƙi mai haske, wanda ake kira β-2-microglobulin; β2M) 17,18 .Sakamakon barga MHC-I trimers ana jigilar su zuwa saman tantanin halitta, inda suke gabatar da peptides na ciki zuwa ƙwayoyin CD8+ T (kwayoyin cytotoxic T) 17.Kwayoyin T suna gane kuma suna lalata waɗannan ƙwayoyin cuta da ƙwayoyin cuta waɗanda ke ɗauke da takamaiman antigen.Sabili da haka, ƙwayoyin cuta da ƙwayoyin ƙwayoyin cuta sukan hana tsarin gabatar da antigen don guje wa sa ido na rigakafi.Bugu da ƙari, MHC-I yana raguwa a cikin 40-90% na ciwace-ciwacen ɗan adam kuma galibi ana danganta shi da ƙarancin tsinkaye19.
Kwayoyin halittar da ke da hannu wajen ba da amsa ga ƙwayoyin cuta dole ne su canza da sauri tsakanin yanayin hutu da yanayin rubutun aiki.Sabili da haka, yawancin sunadaran salula suna tsammanin za su shiga cikin mayar da martani ga babban bukatar IFN a cikin gajeren lokaci, ciki har da gyare-gyare da gyare-gyare na chromatin 20,21 mai gabatarwa.Yawancin karatu sun mayar da hankali kan gano ainihin abokan hulɗar furotin ISG a gaban IFN.Yawancin nazarin proteomic da transcriptomic a cikin tsarin sel na ƙirar ƙira sun haɓaka tasirin IFN akan yanayin salon salula.Duk da haka, duk da haɓakar fahimtar abubuwan da suka haifar da interferon, har yanzu mun san kadan game da shigar ISGs.Idan aka yi la’akari da sarƙaƙƙiya da abubuwan da suka dogara da lokaci na siginar interferon, tambayoyi guda biyu sun taso: (i) shin zai yiwu a daidaitawa da kuma damke abubuwan gina jiki da yawa waɗanda ke cikin saurin sigina, kuma (ii) za a iya tsara waɗannan hulɗar zuwa sararin 3D?
Don magance waɗannan batutuwa, mun aiwatar da disuccinimide suberate-mediated chemical cross-linking (DSS) tare da mass spectrometry (CLMS) don nazarin cibiyar sadarwar furotin da ta haifar da IFNa da haɓakarsa.DSS tana ƙara haɗin haɗin gwiwa tsakanin madaidaitan ragowar sunadaran da/ko rukunin furotin a cikin vivo.Binciken MS na gaba yana bayyana takamaiman rukunin yanar gizo masu alaƙa waɗanda ke nuna kusancin sararin samaniya na yankuna a cikin wani takamaiman furotin, wanda ake kira haɗin kai na ciki, ko sassan cikin rukunin furotin, da ake kira alaƙa.Ta yin amfani da wannan hanyar, mun gano sabbin abubuwan gina jiki-gina jiki da yawa da kuma hanyoyin sadarwar mu'amalar furotin da ke haifar da interferon.Ta ƙarin gwada wani ɓangaren waɗannan sabbin hulɗar, mun nuna cewa H2BFS (H2B-nau'in histone FS; daga baya ake kira H2B) da MDN1 suna aiki azaman abokan haɗin gwiwa don HLA-A.
Kwayoyin Flo-1 suna ɗaya daga cikin mafi kyawun sanannun samfuran in vitro na adenocarcinoma na esophageal yayin da suke kwaikwayi mahimman fasali na ciwace-ciwacen ƙwayar cuta22,23.Duk da haka, ba duk ciwace-ciwacen ƙwayoyi ba ne na rigakafi, kuma don sanin ko ƙwayoyin Flo-1 sun amsa maganin interferon, mun bi da kwayoyin Flo-1 tare da 10 ng / ml IFNα na 72 hours.Kwayoyin Flo-1 sun nuna farkon farawa na pSTAT1 da IRF1, farawa 2 hours bayan jiyya da ci gaba don 72 hours, tare da raguwa mai dogara da lokaci a cikin matakan tsaye na IRF1 (Figure 1A).ISGs (MX1, IFITM1, OAS1/2, da ISG15) an samo su da ƙarfi sosai bayan sa'o'i 6, suna kwaikwayon martani na tsakiyar da ƙarshen lokaci zuwa IFNα (Hoto 1A).Tare, waɗannan bayanan sun nuna cewa ana iya amfani da wannan ƙirar salula don nazarin martanin interferon.
Bambance-bambancen maganganun furotin a cikin ƙwayoyin Flo-1 bayan jiyya na IFNα.(A) Maganin furotin a cikin ƙwayoyin Flo-1 da aka bi da su tare da 10 ng/ml IFNα don 2, 6, 24, 48 da 72 hours an bincikar su ta hanyar immunoblot ta amfani da kwayoyin ISG da aka nuna.(B) Coomassie shuɗi mai launin SDS-PAGE gels na duka tsantsar tantanin halitta bayan haɗe-haɗe tare da DSS don lokuta da yawa da aka nuna.(C) Wakilin immunoblot yayi nazari tare da p53 (DO-1) antibody daga samfurori iri ɗaya don tantance matakin haɗin haɗin furotin.
Don ɗaukar yanayin mu'amalar furotin a wurin, mun yi amfani da DSS, wakili mai haɗin giciye da aka yi amfani da shi sosai saboda yawan iyawarsa da ɗan gajeren lokacin amsawa.Mafi guntu lokacin amsawa yana taimakawa hana samuwar manyan tarukan sunadaran da ke da alaƙa, ta haka ne ke riƙe da kwanciyar hankali na crosslinker.Don tantance mafi kyawun maida hankali na DSS da guje wa wuce gona da iri, mun fara fallasa sel zuwa 5, 2.5, da 1 mM DSS na mintuna 5, 10, 5, da 30, bi da bi, kuma mun bincika lysates ta hanyar SDS-PAGE mai tabo da Coomassie. (Ba a nuna bayanan ba).Kwayoyin lysates sun bayyana suna da alaƙa da haɗin kai a mafi ƙanƙanci kuma a mafi ƙarancin lokaci.Saboda haka, DSS an kidaya shi zuwa 1, 0.5, da 0.1 mM sama da mintuna 5 (Hoto na 1B).An lura da mafi kyawun haɗin kai tare da 0.5 mM DSS na mintuna 5, kuma an zaɓi waɗannan yanayi don ƙwayoyin da aka yi da IFNα.Bugu da kari, Hoto na 1C yana nuna ɓangarorin Yamma da aka yi ta amfani da antibody p53 (DO-1) don tantance matakin haɗin gina jiki.
Kwayoyin Flo-1 an bi da su tare da 10 ng / ml IFNα don 24 hours kafin ƙara da crosslinker.Kwayoyin da ke da alaƙa sun kasance daga baya lysed ta hanyar proteolysis mataki biyu kuma ana sarrafa sunadaran ta FASP (Fig. 2)24,25.An yi nazari akan peptides tryptic masu alaƙa da haɗin gwiwa ta hanyar spectrometry na taro (Fig. 2).Ana daidaita siginar MS/MS zuwa jerin furotin kuma an ƙididdige su tare da MaxQuant26,27.An gano peptides masu haɗin giciye daga abubuwan da aka samo ta hanyar amfani da shirin SIM-XL, kuma an haɗa mahaɗan guda ɗaya a cikin hanyar sadarwa mai rikitarwa ta hanyar amfani da bututun software na buɗaɗɗen software na xQuest28 da SIM-XL29 (Fig. 2).SIM-XL yana gano hulɗar furotin-protein, sarƙoƙi na ciki da sarƙoƙi guda ɗaya a cikin gaurayawar furotin mai sauƙi ko hadaddun kuma yana ba da rubutun don hango ma'amala a cikin tsarin furotin.Bugu da ƙari, yana ƙididdige kowane juzu'in giciye azaman ƙimar ID bisa ga ingancin bakan MS/MS29.An gano hulɗar furotin-gina-gini da yawa da za a iya dogara da su da kuma hadaddun, kuma an ci gaba da bincika sabon tsarin hulɗar ta hanyar amfani da haɗin gwiwar rigakafi da kuma sauye-sauye na gyare-gyare ta hanyar amfani da kwayoyin halitta (MD) samfurin (Fig. 2) 30, 31.
Bayanin tsari na hanyar CLMS.Kwayoyin Flo-1 an bi da su tare da 10 ng / ml IFNα na tsawon sa'o'i 24 sannan a cikin haɗin gwiwar haɗin gwiwar gina jiki ta hanyar amfani da DSS tare da sel lysis da trypsinization.An yi nazarin samfurori masu alaƙa ta hanyar amfani da na'ura mai kwakwalwa ta Orbitrap kuma an ƙara samfurin don rarrabuwa na peptide precursors a lokacin LC-MS/MS.An gano peptides guda biyu masu alaƙa daga sifofin da aka samu ta amfani da Injin Gane Spectrum na shirin Crosslinked Peptides (SIM-XL), kuma duk mahadi an haɗa su cikin hadaddun cibiyar sadarwa ta amfani da bututun lissafi.Tace ƙananan haɗin gwiwa dangane da ƙimar ƙimar ƙimar ƙarya (FDR).An kara tabbatar da wasu sabbin hulɗar furotin da furotin mai ƙarfi ta hanyar amfani da haɗin gwiwar rigakafi, kuma an bincika sauye-sauyen yanayi a cikin rukunin ta amfani da ƙirar ƙwayoyin cuta (MD).
An gano jimlar ~ 30,500 da ~ 28,500 peptides ta amfani da MaxQuant a cikin samfuran IFNα marasa ƙarfi da ƙarfafawa, bi da bi (Ƙarin Teburin S1, Fig. 3A).Rarraba tsawon peptide a cikin lokuta biyu ya nuna mafi girman adadin peptides mafi girma, yana nuna kasancewar peptides masu haɗin gwiwa (Fig. 3B, C).Bugu da ƙari, yawancin peptides mafi girma sun kasance a cikin 40-55 a cikin samfurori na IFNα (Fig. 3C).Taswirar sunadaran akan ƙarfin log2 ya nuna cewa sunadaran da ke motsa interferon sun fi yawa idan aka kwatanta da samfuran da ba a kula da su ba, gami da MX1, IFIT1/3, OAS2/3, DDX58, da HLA-F (Hoto 3D).Binciken hanyoyin da ake amfani da su don sunadaran sunadaran fiye da sau uku waɗanda aka wadatar da su don mayar da martani ga jiyya na IFNα ta yin amfani da bayanan hanyar Reactome ya nuna cewa MHC-I-mediated antigen gabatar da aiki shine hanya mafi rinjaye (Figure 3E).Daidai da rahotannin da suka gabata, martanin antiviral da OAS da ISG15 suka yi tare da IFNα / β da siginar cytokine sun kasance daga cikin hanyoyin da aka kunna.Bugu da ƙari, lysine- da serine-takamaiman furotin na giciye an gano su daga asali na MS/MS da aka samo ta amfani da SIM-XL.Wani bincike na baya-bayan nan ya ba da rahoton ISG 104 da ke tattare da ƙwayoyin cuta 20 daga nau'ikan ƙwayoyin cuta guda 9 ta hanyar nazarin meta-bincike na nazarin yawan adadin ISG guda ɗaya a cikin nau'ikan tantanin halitta 5.Koyaya, don shawo kan iyakokin ƙididdigewa na tantance manyan bayanan bayanai, mun fara da ƙaramin saitin bayanai don bincika yiwuwar hulɗar tsakanin jerin kwayoyin halittar IRDS da Padaria et al., mafi yawansu ISGs ne.
Gano nau'ikan sunadaran da aka bayyana daban-daban masu alaƙa don amsawa ga IFNα (bayanin da aka samu daga MaxQuant).(A) zane na Venn wanda ke wakiltar adadin peptides na yau da kullun da aka gano a cikin IFNα14 da aka yi wa magani da samfuran Flo-1 da ba a kula da su ba.Rarraba tsawon Peptide na samfuran da ba a kula da su ba (B) da IFNα da aka yi wa magani (C) samfurori masu haɗin kai.(D) Taswirar zafi mai wakiltar log2 (ƙarfin LFQ) tsakanin ƙwayoyin Flo-1 da ba a kula da su ba da IFNα14.Ƙungiyar hagu tana nuna sunadaran da aka fi kunnawa a gaban IFNα.(E) Histogram wanda ke wakiltar manyan hanyoyin haɓaka 20 bayan jiyya na IFNα.Bayanan bayanan hanyar Reactome yayi nazarin canje-canje fiye da sau huɗu a cikin furotin masu amsa IFNa.
Interferon-mai shiga tsakani na ISG yana da kyau a rubuce, amma a matakin kwayoyin ba a fahimta da kyau yadda waɗannan sunadaran ke ƙarewa a cikin kewayon ayyukan ilimin halitta.Mun bincika hulɗar furotin tare da babban ƙarfin gwiwa tsakanin sanannun ISGs.Abin sha'awa, mun gano hanyar sadarwa ciki har da MX1, USP18, ROBO1, OAS3, da kuma STAT1 sunadaran da ke samar da babban hadaddun amsa ga maganin IFNα (Hoto 4, Table S2) 32,33,34.Mafi mahimmanci, an samo waɗannan hulɗar a cikin duk nau'i uku da aka bi da su tare da IFNα kuma ba a samo su a cikin samfurori da ba a kula da su ba, suna nuna cewa an kafa su musamman don amsawa ga maganin IFNα.An san cewa STAT1 yana sarrafa bayanin waɗannan ISGs a rubuce, amma ba a yi nazarin hulɗar ta da ISGs a matakin furotin ba.Tsarin crystal na STAT1 ya nuna cewa yankin sa na helical (CCD) ba shi da hannu a cikin hulɗa tare da DNA ko protomers yayin samuwar dimers35.Wadannan α-helices suna samar da tsarin helix na helical wanda ke ba da wuri mai mahimmanci na hydrophilic don hulɗar da ke faruwa 35.A cikin bayanan mu na CLMS, mun lura cewa yawancin hulɗar tare da STAT1 sun faru a cikin yankin SH2 da ke gaba da CCD, yankin mahaɗa, ko wutsiya ta C-tashar (raguwar 700-708) (Hoto 4A).Wani binciken da ya gabata ya ruwaito cewa USP18 yana ɗaure zuwa CCD da DNA-binding domain (DBD) na STAT2 kuma an ɗauke shi zuwa sashin nau'in mai karɓa na I IFNAR2 don daidaitawa da hana nau'in I interferon siginar 24.Bayananmu kuma sun nuna cewa yankin catalytic na USP18 yana hulɗa tare da STAT1 DBD (Hoto 4A,D), yana nuna cewa duka STAT1 da STAT2 na iya taka rawa wajen jawo USP18 zuwa IFNAR2.
Protein-protein ISG cibiyar sadarwar da aka gano a cikin sel masu alaƙa da aka yi da IFNα.(A) makircin hulɗar 2D yana nuna hulɗar sunadaran-gina jiki (wanda aka ƙirƙira a cikin shirin SIM-XL), tare da layin da ke wakiltar hulɗar intermolecular (crosslink cutoff saita zuwa 3.5).Domains na ainihi daban-daban ana yiwa alama ta launi32: yankin MX1, Dynamin_N (73-249), Dynamin_M (259-547), da GED (569-660).Yankunan OAS3: OAS1_C (160-344), OAS1_C (559-745), NTP_transf_2 (780-872), da OAS1_C (903-108).Domain ROBO1, Ig_3 (67-151), I-set (170-258), I-set (262-347), Ig_3 (350-432), Ig_3 (454-529), fn3 (562-646), fn3 (678-758) da fn3 (777-864).Filayen STAT1: STAT_int (2-120), STAT_alpha (143-309), STAT_bind (321-458), SH2 (573-657), da STAT1_TAZ2bind (715-739).(B) Mai kallon madauwari na sunadaran da aka haɗe (MX1, UBP18, OAS3, ROBO1, da STAT1) tare da hulɗa da hulɗar da aka yi wa lakabi da shuɗi da ja, bi da bi.An saita madaidaicin hanyar haɗin kai a 3.5.Maƙallan ɗigo suna nuna wuraren hulɗar STAT1 tare da MX1 (C), USP18 (D), ROBO1 (E), da OAS3 (F), da kuma wuraren hulɗar K ko S tsakanin peptides biyu.A cikin adadi, an saita maƙiyan giciye zuwa 3.0.(G) Shafukan hulɗa daban-daban tsakanin STAT1 da OAS3 DI yankuna da aka sanya su akan sifofin sunadaran su a cikin PyMol (Tsarin zane-zane na PyMOL, sigar 2.0 Schrödinger, LLC.);STAT1 (pdb id: 1bf533) da OAS3 (pdb id: 4s3n34).) shirin.
An kwatanta nau'i biyu na USP18 a cikin mutane, cikakken sunadaran sunadaran da ke da yawa a cikin tsakiya, da kuma isoform ba tare da yankin N-terminal ba, USP18-sf, wanda aka rarraba a ko'ina a cikin cytoplasm da tsakiya 36.Bugu da ƙari, an annabta N-terminus ba shi da tsari kuma baya buƙatar aikin isopeptidase ko ɗaurin ISG1537.Yawancin hulɗar da aka gano a cikin bincikenmu suna cikin N-terminus na furotin, yana nuna cewa waɗannan hulɗar sun haɗa da USP18 cikakke (Figure 4A, D) kuma don haka yana iya faruwa a cikin tsakiya.Haka kuma, bayananmu sun kuma nuna cewa N-terminus ya ƙware don hulɗar furotin-zuwa-gina jiki.Wurin daurin IFNAR2 yana tsakanin ragowar 312-368, kuma musamman, babu wani sunadaran da ke cikin hadaddun da ke ɗaure zuwa wannan yanki (Fig. 4A) 37,38 .Waɗannan bayanan da aka haɗa tare suna nuna cewa yankin dauri na IFNAR2 yana amfani da furotin mai karɓa kaɗai.Bugu da ƙari, kawai OAS3 da ROBO1 an samo su da alaƙa da yankunan sama na N-terminus da IFNAR2 dauri site (Hoto 4A).
ROBO1 na cikin dangin immunoglobulin (Ig) na ƙwayoyin siginar transmembrane kuma ya ƙunshi yankuna Ig guda biyar da yankuna fibronectin (Fn) guda uku a cikin yankin extracellular.Wadannan yankuna na waje suna biye da yankin membrane-proximal da helix transmembrane guda ɗaya 39. Yankin intracellular da ba a tsara shi ba yana samuwa a cikin C-terminus kuma yana ƙunshe da jerin abubuwan da aka kiyaye su wanda ke daidaita tasirin furotin mai tasiri39.Yankin da ya tashi daga amino acid ~ 1100 zuwa 1600 galibi yana da matsala.Mun gano cewa MX1 yana hulɗa tare da ROBO1 ta hanyar Ig, Fn, da kuma yankunan intracellular, yayin da yawancin hulɗar da STAT1 ke faruwa a tsakanin CCD, mahaɗin yanki, da kuma C-terminus na ROBO1 (Fig. 4A, E).A gefe guda, an rarraba hulɗar tare da DI, DIII, da OAS3 yankuna masu haɗin gwiwa a ko'ina cikin furotin ROBO1 (Fig. 4A).
Iyalin furotin na oligoadenylate synthase (OAS) suna karɓa kuma suna ɗaure RNA (dsRNA) na ciki na ciki, yana fuskantar canje-canje masu daidaituwa, kuma yana haɗa oligoadenylates 2′,5′-linked (2-5 As) 40.An gano cewa a cikin OAS guda uku, OAS3 yana nuna mafi girman kusanci ga dsRNA kuma yana haɗa mafi ƙarancin adadin 2-5 Kamar yadda, wanda zai iya kunna RNase L kuma ta haka yana iyakance kwafin hoto na 41.Iyalin OAS sun ƙunshi polymerase beta (pol-β) -kamar yankunan transferase na nucleotide.Binciken da ya gabata ya nuna cewa aikin catalytic na yankin C-terminal (DIII) ya dogara da yankin dsRNA-binding (DI), wanda ake buƙata don kunna OAS342.Mun lura cewa yankunan DI da DII na OAS3 suna hulɗa tare da CCD da ƙananan yanki tsakanin SH2 da STAT1 TAD (Hoto 4A, F).Rufe shafuka daban-daban na ƙetare akan tsarin furotin ya nuna hulɗar tsakanin madauki na β-sheet da DBD STAT1 madauki da buɗaɗɗen aljihu ko rami da aka kafa ta ragowar 60-75 a cikin yankin DI na OAS3 (Fig. 4G).Matsakaicin sunadaran a cikin hadaddun kuma ya nuna cewa babu wani hulɗa tare da OAS3 da ya tsoma baki tare da ikon ɗaure DNA na yankin DI (Fig. S1A).Bugu da ƙari, yankin N-terminal na GTPase MX1 yana hulɗa da yawa tare da yankunan DI da DIII na OAS3 (Fig. 4A).Mun kuma lura da hulɗar tsakanin OAS1 da MX1 a cikin duka uku na IFNα da aka yi maganin maimaitawa, inda yankin OAS1 guda ɗaya (kuma yana aiki mai mahimmanci) ya yi hulɗa tare da dukkanin yankunan MX1 guda uku (Figure S2A, B).
Sunadaran MX wani ɓangare ne na babban iyali na dynein-kamar GTPases waɗanda ke ƙunshe da yanki na N-terminal GTPase wanda ke ɗaure da hydrolyzes GTP, yanki na tsakiya wanda ke daidaita haɗin kai, da kuma C-terminal leucine zipper wanda ke aiki a matsayin GTPase (LZ). ).mai tasiri yankin yanki25,43.MX1 yana ɗaure zuwa ƙananan ƙwayoyin cuta na polymerases don toshe fassarar kwayar cutar kwayar cutar kwayar cutar kwayar cutar kwayar cuta ta gene43.Wani yisti da aka ba da rahoto a baya-tsalle-tsalle-tsalle ya nuna cewa MX1 mai haɗin gwiwa na PIAS1 ya hana STAT1-matsakaicin ƙaddamar da kwayar halitta ta hanyar toshe ayyukan ɗaurin DNA kuma yana da SUMO E344,45 ayyukan ligase.Anan, mun nuna cewa MX1 yana ɗaure zuwa STAT1 (Hoto 4C, D), duk da haka yadda wannan hulɗar ke shafar STAT1-mediated gene activation a mayar da martani ga IFNa yana buƙatar ƙarin nazari.Bugu da ƙari, mun kuma gano cewa MX1 ya yi hulɗa tare da IFIT3 da DDX60 a cikin duka uku na IFNα-magance maimaita (Fig. S2C).
DDX60 shine helicase na cytoplasmic wanda aka haifar da IFN wanda a baya aka ruwaito yana taka rawa a RIG-I mai cin gashin kansa na lalata RNA46.Yana hulɗa tare da RIG-I kuma yana kunna siginar sa a cikin ƙayyadaddun ƙayyadaddun ƙayyadaddun ligand 46. DDX60 ya ƙunshi yanki na DEXD / H-Box da yanki na helicase na C-terminal wanda ke ɗaure RNA da DNA47.Yawancin hulɗar sa tare da MX1 da IFIT3 suna faruwa a cikin dogon N- da C-terminal yankuna ba tare da yankunan canonical ko motifs (Fig. S2E, F).Duk da haka, MX1 kuma yana da alaƙa da DEXD/H-Box helicase domain (Fig. S2E).Sunadaran dangin IFIT suna da kwafin tandem na wani keɓantaccen tsarin helix-turn-helix da ake kira tetrapeptide repeat (TPR).An samo IFIT3 a matsayin mai daidaitawa mai kyau na RIG-I sigina kuma saboda haka wani ɓangare na hadaddun MAVS.Haɗe tare, bayananmu sun nuna cewa IFIT3 da DDX60 suna hulɗa da farko a cikin yankin tsakanin TPR 3-6 na IFIT3 kuma yana iya taka rawa a cikin siginar RIG-I / MAVS (Fig. S2F).
Ganin cewa yin gwajin gabaɗayan proteome yana da ƙarfi cikin lissafi, sannan muka bincika duk bayanan UniProt na ɗan adam don kasancewar ɗaya daga cikin maimaitawar IFNα.A cikin wannan kwafin, mun sami amintattun hanyoyin sadarwar hulɗa don HLA-A.Binciken hanyoyin gina jiki da aka gano ta hanyar MS/MS spectra ya nuna cewa MHC-I-based antigen aiki da gabatarwa shine babbar hanyar da interferon ta haifar (Fig. 3D).Sabili da haka, mun mayar da hankali kan nazarin hulɗar furotin na kwayoyin MHC-I tare da babban matsayi na amincewa a duk samfurori masu alaƙa.HLA ya ƙunshi yanki na α1, α2 da α3 da sarƙoƙi masu haske, kuma microglobulin β2 (β2m) furotin ne na chaperone na dindindin49.Da zarar an taru a cikin endoplasmic reticulum, HLA ba shi da kwanciyar hankali idan babu peptide ligands50.An samar da tsagi mai ɗaurin peptide ta hanyar polymorphic sosai da α1 da α2 waɗanda ba a tsara su ba a cikin nau'in nau'in peptide da ƙananan yanki na polymorphic α351.A gaban IFNα, mun gano nau'ikan HLA-A guda biyu: ɗayan yana hulɗa da HMGA1 da H2B (Hoto 5, Table S3) kuma ɗayan yana hulɗa tare da MDN1, LRCH4 da H2B (Hoto 6).
IFNα yana haifar da hanyar sadarwar HLA-A tare da H2B (H2BFS) da HMGA1.(A) 2D mãkirci (wanda aka ƙirƙira a cikin software na SIM-XL) yana nuna nau'ikan hulɗa a cikin hadaddun H2B-HLA-A-HMGA1: interlink (blue), interlink (ja) da mahaɗin guda ɗaya (black)..Domains na ainihi daban-daban sune masu launi32: H2B (histone; 2-102) da MHC-I (MHC_1; 25-203, rukunin C1; 210-290 da MHC_I_C; 337-364).An saita madaidaicin hanyar haɗin kai a 3.5.Maƙallan ɗigo suna nuna wuraren hulɗar HLA-A tare da H2B (B) da HMGA1 (C), da kuma wuraren hulɗar K ko S tsakanin peptides biyu.A cikin adadi, an saita maƙiyan giciye zuwa 3.0.(D) Dangantaka tsakanin sunadaran da aka nuna a cikin sifofin H2B, HLA-A, da HMGA1 a cikin shirin PyMOL.An tsara waɗannan sifofin ta amfani da uwar garken Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) da tsarin samfuri na sunadaran H2B, HLA-A da HMGA1 sune 1kx552, 1kj349 da 2eze55, bi da bi.
IFNα yana haifar da hanyar sadarwar HLA-A tare da H2B (H2BFS), MDN1 da LRCH4.(A) Intramolecular (ja) da intermolecular (blue) crosslinks da aka gabatar akan taswirar mu'amala ta 2D (wanda aka ƙirƙira a cikin software na SIM-XL) tare da MDN1 wakilta azaman da'irar.An saita madaidaicin hanyar haɗin kai a 3.5.Domains na ainihi daban-daban sune masu launi32: H2B (histone; 2-102), MHC-I (MHC_1; 25-203, rukunin C1; 210-290 da MHC_I_C; 337-364) da LRCH4 (LRR_8 (68-126), LRR_8 (137-194) da CH (535-641)).(B) Dangantaka tsakanin sunadaran da aka nuna a cikin sifofin H2B, HLA-A, LRCH4, da MDN1 sunadaran a cikin shirin PyMOL.An tsara waɗannan tsarin ta amfani da uwar garken Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) tare da tsarin samfuri 1kx552, 1kj349, 6hlu62 da 6i2665 don sunadaran H2B, HLA-A, LRCH4 da MDN1, bi da bi.Maƙallan ɗigo suna nuna wuraren hulɗar K ko S don HLA-A tare da H2B (C), LRCH4 (D), da MDN1 (E).Don filaye, an saita makin giciye zuwa 3.0.
Baya ga kiyaye mutuncin kwayoyin halitta, histone H2B kuma yana da hannu a cikin ƙa'idar rubutun.Sunadaran H2B ya ƙunshi babban yanki na histone (HFD) wanda aka kafa ta uku-helices waɗanda aka raba ta madaukai da wutsiya ta C-terminal 41,52.Yawancin hulɗar tare da H2B yana faruwa a cikin α1 helix, wanda ke ba da trimerization tare da HFD heterodimer (Fig. 5A, B).Kodayake lysins suna da hannu a ɗaurin DNA, wasu lysins kuma madadin acetylation ne ko rukunin methylation.Misali, ragowar K43, K46, da K57 na H2B ba su da hannu cikin ɗaurin DNA kai tsaye, amma hari ne na gyare-gyaren rubutu daban-daban53.Hakazalika, ragowar K44, K47, da K57 a cikin H2B na iya taka wani matsayi na dabam a gaban IFNα, ciki har da hulɗa tare da sauran sunadaran (Fig. 5A, B).Bugu da ƙari, extrachromosomal histone H2B yana kunna amsawar rigakafi a cikin nau'ikan tantanin halitta daban-daban, yana aiki azaman firikwensin cytosolic don gano gutsuttsuran DNA (dsDNA) guda biyu waɗanda aka samo daga masu kamuwa da cuta ko ƙwayoyin da suka lalace54.A gaban ƙwayoyin cuta na DNA, raguwar H2B ya hana samar da IFN-β da STAT154 phosphorylation.H2B kuma an san shi don motsawa da fita daga tsakiya cikin sauri fiye da sauran tarihin tarihi54.An kuma lura da hulɗar H2B tare da MDN1 da LRCH4 a cikin zaɓaɓɓun samfuran da ba a kula da su ba.Mun gano cewa HLA-A ta yi hulɗa tare da H2B a cikin dukkanin samfuran IFNa guda uku da aka yi wa magani kuma a cikin samfurin maimaita ba tare da magani ba.Waɗannan bayanan suna nuna rawar H2B a cikin madadin aikin physiological wanda ba shi da ƙa'idar rubutu.
HMGA1 (ƙungiyar motsa jiki mai girma AT-Hook 1), ƙaramin nucleoprotein mai wadata a cikin amino acid masu haɓaka cuta, an gano shi tare da HLA-A.Yana da wutsiyar C-terminal acidic da DBD guda uku daban-daban da ake kira AT hooks saboda suna ɗaure ga ƙaramin tsagi na yanki mai arzikin AT a dsDNA55,56.Wannan ɗaurin yana haifar da DNA ta lanƙwasa ko daidaitawa, yana barin abubuwan rubutun canonical don samun damar jerin ijma'i.An yi imanin wutsiya ta C-terminal tana da hannu a cikin hulɗar sunadaran-gina jiki da kuma ɗaukar abubuwan da aka rubuta, tun da C-terminal deletion mutants ba su iya fara rubutawa57.Bugu da ƙari, wannan yanki ya ƙunshi wurare da yawa da aka adana na phosphorylation waɗanda aka san su don kinases 58 .Mun lura da hulɗar HLA-A da H2B tare da HMGA1 a waje da yankin C-terminal, yana nuna cewa an fi amfani da yankin C-terminal don ɗaure factor factor (Fig. 5A, C).Sunadaran HMGA suna gasa tare da histone H1 don ɗaure zuwa adaftar DNA, ta haka ƙara samun dama57.Hakazalika, da alama HMGA yana hulɗa da histone H2B tare da mahaɗan DNA a cikin gasa tare da histone H1.HMGB1 yana haifar da bayyanar HLA-A, -B, da -C a cikin ƙwayoyin dendritic, wanda ke haifar da kunna su59, amma hulɗar tsakanin HMG da HLA ba a ba da rahoto a baya ba.Mun gano cewa HMGA1 yana hulɗa tare da yankunan α1 da α3 na HLA-A, tare da yawancin hulɗar da ke waje da 3 DBD (Hoto 5A,C).A cikin hannayenmu, an gano HLA-A a cikin tsakiya (bayanan da ba a nuna ba), kuma an ba da cewa H2B da HMGA1 suna cikin tsakiya, wannan hulɗar yana iya faruwa a cikin tsakiya.Takamaiman abubuwan da aka auna tsakanin H2B, HLA-A, da HMGA1 ana nuna su a hoto na 5D.
Yawancin hulɗar HLA-A tare da wasu sunadaran suna faruwa a cikin yankunan α1 da α2 da kuma yankin C-terminal maras kyau (Fig. 6).A cikin ɗaya daga cikin waɗannan misalan, mun gano cewa HLA-A yana hulɗa tare da wutsiyar N-terminal na LRCH4 (Hoto 6A,D).LRCH4 yana sarrafa kunnawar TLR4 da shigar da LPS cytokine, ta haka ne ke daidaita martanin rigakafi na asali60,61.Yana da furotin mai gina jiki tare da maimaitawar leucine tara (LRRs) da kuma calmodulin (CH) homology motif a cikin ectodomain ta, sannan kuma wani yanki na transmembrane (TMD) 60, 62.An ba da rahoton yankunan CH don yin hulɗar haɗin gwiwar furotin-protein 60.Tsakanin amino acid kusan 300 tsakanin yankunan LRR da CH yana da ɗan sauƙi amma ba su da ƙarfi.Dangane da aikin yankunan da ba su da kyau a matsayin masu shiga tsakani na hanyoyin sadarwa na furotin-protein da kuma jigilar vesicular 63, mun gano cewa yawancin haɗin gwiwar sunadaran suna faruwa a yankunan da ba su da kyau.An rarraba hulɗar tare da MDN1 a cikin tsawon tsawon furotin, ciki har da yankunan LRR1, LRR6, CH, da yankunan bazuwar, yayin da H2B ya fi dacewa da yankin CH (Fig. 6A, B).Musamman ma, babu ɗayan hulɗar da ya haɗa da TMJ, yana nuna ƙayyadaddun tsarin CLMS (Hoto 6A, B).
An kuma gano MDN1 a matsayin ɓangare na hanyar sadarwar furotin HLA-A (Hoto 6A).Yana cikin dangin AAA na sunadaran (ATPases masu alaƙa da ayyuka daban-daban).Wannan yanki ɗaya ne na N-terminal AAA wanda ke tsara zuwa zoben hexameric kuma yana cire ma'aunin taro daga 60S 64 ribosomal subunit.ya bayyana yana kama da dynein64,65,66.Bugu da kari, yankin mai arzikin Asp/Glu yana biye da yankin MIDAS (shafin dogara da karfe).Saboda girman girman MDN1 (kimanin amino acid 5600) da ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun sunadaran gina jiki, an san kaɗan game da tsarinsa da aikinsa a cikin mutane.Mun gano HLA-A, H2B, da LRCH4 a matsayin abokan haɗin gwiwa na MDN1 kuma mun bayyana yanayin su azaman rukunin furotin a cikin PyMol (Fig. 6A, B).Waɗannan sunadaran guda uku suna hulɗa tare da yankin AAA, yanki mai kama da dynein, da yuwuwar yankin MIDAS MDN1.A cikin rahoton da ya gabata, tsarkakewar sunadaran koto sun gano MDN1 a matsayin furotin da ke da alaƙa da histone H2B67.Bugu da ƙari, wani binciken da aka yi kwanan nan ya kuma ba da rahoton hulɗar tsakanin MDN da HLA-B a cikin kwayoyin HCT116 ta amfani da nau'i mai tsabta mai tsabta, yana tallafawa bincikenmu68.Gano wannan hadaddun a cikin samfuran da aka yi wa IFNα suna nuna rawar MDN1 a cikin siginar interferon.
Saboda kwayoyin halittar HLA suna da yawa polymorphic, mun fitar da jerin abubuwan karanta taswirar HLA-A, -B, da -C daga bayanan jerin RNA na ƙwayoyin Flo-1 (bayanan da ba a nuna ba).Jerin Peptide wanda ya yi daidai da karatun jeri ya bayyana manyan bambance-bambance tsakanin HLA-A, -B, da -C a cikin yankuna inda peptides masu alaƙa da giciye ke cikin HLA-A (Hoto S3).Bugu da ƙari, ba mu lura da haɗin gwiwar furotin-zuwa-protein na ƙwayoyin HLA-B/C tare da sunadaran H2B/HMGA1/MDN1/LRCH4 ba.Wannan yana nuna cewa hulɗar sunadaran da aka samo tsakanin HLA-A, MDN1, LRCH1 da HMGA1 shine takamaiman HLA-A.Bugu da ƙari, nazarin proteomic na samfuran da ba a haɗa su ba (Table S4) ya nuna cewa HLA-A yana da mafi girman ɗaukar hoto idan aka kwatanta da HLA-B ko HLA-C.Abubuwan peptides da aka gano don HLA-A sun kasance masu ƙarfi a cikin duka samfuran IFNa da ba a kula da su ba.
Don tabbatar da cewa ma'amalar da aka gano a nan ba ta kasance saboda ƙayyadaddun haɗin kai na sunadaran sunadarai guda biyu a cikin kusancin sararin samaniya ba, mun ƙara tabbatar da sabbin abubuwan hulɗar HLA-A guda biyu ta hanyar yin gwaje-gwajen rigakafin rigakafi.An gano hulɗar HLA-A tare da MDN1 na ƙarshe da H2B a cikin duka IFNα da aka yi wa magani da ƙwayoyin Flo-1 marasa magani (Hoto 7, Hoto S4).Mun tabbatar da cewa H2B ya kama HLA-A a cikin maganin rigakafi kuma wannan ƙungiyar ta kasance saboda maganin IFNα tun lokacin da HLA-A ba ta nan a cikin samfurori na rigakafi daga kwayoyin da ba a kula da su ba (Figure 7A).Koyaya, bayananmu sun ba da shawarar cewa IFNa ta bambanta tana daidaita HLA-A ɗaurin H2B da MDN1.IFNα yana haifar da haɗin gwiwa tsakanin H2B da HLA-A, amma yana rage haɗin gwiwa tare da MDN1.Mun gano cewa MDN1 yana da alaƙa da HLA-A a cikin sarrafawa, kuma ƙari na IFNα ya rage wannan hulɗar mai zaman kanta ta shigar da MDN1 ta IFNα (Hoto 7B, C).Bugu da ƙari, HLA-A immunoprecipitation kama H2B a cikin kwayoyin A549 (Fig. S4), yana nuna cewa wannan hulɗar ta kasance mai zaman kanta daga nau'in tantanin halitta.Haɗe tare, waɗannan sakamakon suna goyan bayan hulɗar interferon-matsakaici na HLA-A tare da H2B da MDN1.
HLA-A yana haɓaka H2B da MDN1.Wakilin H2B (A) da MDN1 (B) immunoblots an yi rigakafi daga ƙwayoyin Flo-1 da aka yi wa IFNa kuma an bincikar ƙwayoyin rigakafin da aka nuna.An yi amfani da linzamin kwamfuta da zomo IgG azaman iko mara kyau.(C) Matsakaicin adadin (shigarwar) na antigens daban-daban ana nuna su ta hanyar immunoblots da aka yi bincike akan ƙwayoyin rigakafi da aka nuna, an yi amfani da β-actin azaman sarrafa kaya.
An bincika kaddarorin tsarin ɗaya daga cikin hanyoyin sadarwa masu haɗin kai da aka haifar da interferon sosai, H2B-HLA-A-HMGA1.Mun yi amfani da ƙirar ƙirar ƙwayoyin ƙwayoyin cuta a matsayin madadin hanya don fahimtar haɓakar haɓakar sunadaran da ke cikin wannan hadaddun (Hoto 8).Bayanan bayanai daga bayanan CLMS suna ba da shawarar yuwuwar daidaituwa daban-daban na sunadaran H2B, HLA-A, da HMGA1.Sabili da haka, an ƙirƙira waɗannan yuwuwar rukunin gidaje a cikin matsakaicin ƙarfi: H2B-HLA-A, HMGA1-HLA-A, da H2B-HLA-A-HMGA1.Kunshin docking na furotin-protein na farko ta amfani da MOE (Molecular Operating Environment; Chemical Computing Group Inc., Montreal, Quebec, Canada) kunshin ya ba da shawarar yiwuwar daidaitawa da suka bambanta tsakanin waɗannan sunadaran (Fig. 8A).Hange na rukunin furotin na docking ya bayyana hulɗa da yawa da yuwuwar daidaituwa (Hoto 5A, 8).Don haka, ana nuna daidaituwa ɗaya mai yuwuwa a cikin Hoto na 8A (tare da alamar haɗin giciye) kuma an ƙara kimanta ta ta amfani da bututun ƙirar MD.Bugu da ƙari, ɗaurin H2B ko HMGA1 zuwa HLA-A yana nuna mafi girman alaƙar H2B don HLA-A (Fig. 8A).
Matsakaicin daidaituwa na yuwuwar hanyoyin sadarwa tsakanin rukunin H2B (H2BFS) -HLA-A, HMGA1-HLA-A, da H2B-HLA-A-HMGA1.(A) Taswirar hagu taswirar 2D ce (an ƙirƙira a cikin software na SIM-XL) na intramolecular (ja) da tsaka-tsakin tsaka-tsaki (shuɗi) (yanke hanyar haɗi zuwa 3.5).Bugu da kari, ragowar abubuwan haɗin kai da aka gano ana yiwa lakabin akan sigar sunadaran H2B, HLA-A, da HMGA1.Abubuwan da ke da alaƙa na waɗannan sunadaran an fitar da su ta amfani da bututun docking da aka aiwatar a cikin kunshin MOE.Ƙashin hagu na ƙasa yana nuna nau'ikan yuwuwar daidaituwa na rukunin H2B-HLA-A da HMGA1-HLA-A tare da alaƙar ɗaurin furotin-protein daban-daban (GBVI/WSA dG; kcal/mol).(B) Daidaitaccen karkata (RMSD) na matsayin atomic (ban da hydrogen atom) don kowane tsarin gina jiki.(C) Haɗin haɗin furotin-protein hydrogen haɗin gwiwa daga rukunin simulators daban-daban la'akari da takamaiman ma'amala na tsawon lokaci ≥ 10 ns.An saita nisan yanke mai ba da gudummawa-mai karɓa na h-bond zuwa 3.5 Å, kuma an saita kusurwar yanke mai bayarwa-H-mai karɓa zuwa ≥ 160°-180°.(D) Abubuwan da aka lakafta suna yin hulɗar HLA-A sunadaran sunadaran gina jiki tare da abokan aikinsu, wanda ya kai ≥ 20 ns, wanda aka ciro daga rukunin HLA-A-H2B da HLA-A-HMGA1 masu lalata.Tsarin sunadaran suna wakiltar matsakaicin tsari na 100 ns MDS.(E) Ma'amala tsakanin hadaddun HLA-A-H2B da HLA-A-HMGA1 idan aka kwatanta da ma'amalar da H2B-HLA ke bibiyar simulation sama da 100 ns dangane da wurin hulɗar K ko S tsakanin peptides biyun.Complexes /HMGA1-HLA-A/H2B-HLA-A-HMGA1.An saita ƙimar ƙima don kimanta hanyoyin haɗin kai zuwa 3.0, kuma an yi la'akari da takamaiman ma'amala daga MDS ɗaukar ≥ 10 ns.An hango sifofin furotin ta amfani da fakitin BIOVIA Discovery Studio (Dassault Systèmes, BIOVIA Corp., San Diego, CA, Amurka) da Molecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada).
Zaman lafiyar kwayoyin HLA-A na tsawon lokaci (daidaituwar ma'auni; RMSD ko daidaitattun daidaito; RMSF) ya nuna cewa kasancewar H2B ko HMGA1 sunadaran a cikin hadaddun sun daidaita HLA-A (Hoto 8B, Hoto S5).Sunadaran HMGA1 yana ɗaure tam zuwa rukunin B2M na HLA-A, yana haifar da kwanciyar hankali na HLA-A amino acid a cikin hadaddun HLA-A-HMGA1 ko H2B-HLA-A-HMGA1 (Hoto 8B, Hoto S5).musamman, ragowar HLA ~ 60-90 da ~ 180-210 an samo su ba su da sauƙi a gaban H2B (FIG. 8B).H2B da HMGA1 sun nuna mafi kyawun ɗaurin HLA-A a cikin hadaddun H2B-HLA-A-HMGA1 idan aka kwatanta da HLA-A daure zuwa H2B ko HMGA1 kaɗai (Hoto 8C,D; Table S5).Ragowar da ke tattare da haɗin gwiwar hydrogen (MD wanda aka ƙirƙira babban zama ≥ 10 ns) sun yi daidai da wuraren hulɗar CLMS ( ragowar K ko S) a cikin hadaddun, yana nuna cewa hulɗar da CLMS ta gano suna da aminci sosai.Amincewa (Fig. 8E).A cikin ƙirar CLMS da MD, ragowar HLA-A tsakanin kusan 190-210 da kusan 200-220 amino acid an sami su ɗaure H2B da HMGA1, bi da bi (FIG. 8E).
Ma'amalar furotin da sunadaran suna samar da hanyoyin sadarwa masu ƙarfi waɗanda ke daidaita sadarwa ta cikin salula don amsa wasu abubuwan motsa jiki.Saboda da yawa hanyoyin proteomics suna gano canje-canje a cikin gaba ɗaya daidaita matakin furotin, haɓakar hulɗar sunadaran sunadaran suna buƙatar ƙarin kayan aiki don kama hanyoyin haɗin kai, kuma CLMS ɗaya ce irin wannan kayan aikin.Tsarin sigina na interferon shine cibiyar sadarwar cytokine wanda ke ba da damar sel don amsa nau'ikan siginar cututtukan cututtukan muhalli da na ciki, wanda ya ƙare a cikin shigar da sassan sunadaran-inducible interferon.Mun yi amfani da CLMS don tantance ko za a iya gano hulɗar sunadaran sunadaran gina jiki a tsakanin rukunin sunadaran da ke haifar da interferon.Binciken haɗin gwiwar furotin na duniya a cikin ƙirar tantanin halitta Flo-1 mai amsa interferon an yi amfani da shi don kama rukunin furotin.Cire peptides na tryptic daga ƙwayoyin da ba a haɗa su ba da sel masu haɗin gwiwa suna ba da damar ƙidayar peptide, haɓakar hanya, da rarraba tsawon peptide tare da ƙayyadaddun ƙarfin LFQ.Canonical interferon-inducible sunadaran an gano su azaman ingantacciyar kulawa ta ciki, yayin da aka lura da sabbin hanyoyin haɗin gwiwar interferon-inducible na canonical interferon-inducible sunadaran kamar MX1, UP18, OAS3 da STAT1.An bincika fasalulluka iri-iri da mu'amala a wuraren aiki.
An gano hulɗar tsakanin HLA-A, MDN1 da H2B ta hanyar immunoblotting a cikin ƙwayoyin Flo-1 da A549 da aka bi da su kuma ba a kula da su tare da IFNα.Sakamakonmu yana nuna cewa HLA-A yana tattare da H2B a cikin hanyar dogaro da IFNa.Ayyukanmu yana wakiltar hanya mai ban sha'awa don ƙarin bincike na haɗin gwiwa na waɗannan gidaje biyu.Hakanan zai zama mai ban sha'awa don faɗaɗa tsarin CLMS zuwa rukunin layin tantanin halitta don gano ma'amalar furotin mai zaman kanta ta nau'in tantanin halitta.A ƙarshe, mun yi amfani da ƙirar MD a matsayin wata hanya ta dabam don fahimtar yanayin haɓakar sunadaran da ke da hannu a cikin hadaddun H2BFS-HLA-A-HMGA1, waɗanda ke bibiyar magana ta intramolecular da intermolecular.Bayanan bayanai daga bayanan CLMS suna ba da shawarar yuwuwar daidaituwa daban-daban na sunadaran H2BFS, HLA-A, da HMGA1.Yiwuwar daidaituwa daban-daban tsakanin waɗannan rukunin sunadaran gina jiki sun bayyana ma'amala da yawa kama da waɗanda aka gani a cikin bayanan CLMS.Ɗaya daga cikin manyan ƙarfin hanyarmu shine yana ba da damar gano sauƙi na hulɗar kwayoyin halitta masu polymorphic kamar HLA, don haka zai zama mai ban sha'awa don nazarin hulɗar takamaiman sunadaran HLA haplotype waɗanda suke da wuyar nazari.A haɗe, bayananmu sun nuna cewa ana iya amfani da CLMS don faɗaɗa fahimtarmu game da hanyoyin sadarwar sigina da ke haifar da interferon da kuma samar da tushe don nazarin ƙarin hadaddun tsarin tsaka-tsakin ƙwayoyin cuta a cikin ƙananan ƙwayoyin cuta.
An samo ƙwayoyin Flo-1 daga ATCC kuma an kiyaye su a cikin DMEM (Gibco) tare da 1% penicillin / streptomycin (Invitrogen), 10% fetal bovine serum (Gibco) kuma an adana shi a 37 ° C da 5% CO2.Shigarwa.Kwayoyin sun girma zuwa 70-80% haɗuwa kafin a yi musu magani tare da IFNα14 (wanda Edinburgh Protein Production Facility ke ƙera).Duk sauran sinadarai da reagents an sayi su daga Sigma Aldrich sai dai in an lura da su.
Kwayoyin Flo-1 an tsara su a cikin faranti na 6-riji kuma a rana ta gaba an bi da kwayoyin tare da 10 ng / ml IFNα14 don 24 hours zuwa kusan 80% haɗuwa.An wanke sel sau uku tare da PBS kuma an haɗa su tare da DSS da aka shirya (Thermo Fisher Scientific) (narkar da su a DMSO) a cikin PBS don 5 min a 37 ° C. zuwa ƙaddamarwar ƙarshe na 0.5 mM.An maye gurbin halayen haɗin gwiwar DSS da PBS kuma an kashe ragowar DSS ta ƙara 20 mM Tris (pH 8.0) a cikin PBS na mintuna 15 a 37°C.An tattara sel ta hanyar gogewa kuma an tattara su a cikin ƙananan bututu masu ɗaure (Axygen).
An lulluɓe pellet ɗin tantanin halitta tare da 300 µl na buffer urea lysis (8 M urea, 0.1 M Tris, pH 8.5) na 30 min a zazzabi na ɗaki tare da girgiza lokaci-lokaci.An yi duk matakan centrifugation a 14,000 xg a 8 ° C.Centrifuge lysate na minti 10 kuma canja wurin supernatant zuwa sabon bututu.An narkar da sauran abubuwan da suka rage a cikin 150 μl na buffer lysis na biyu (2 M urea, 2% (w / v) SDS (sodium dodecyl sulfate)) na minti 30 ko fiye har sai an sami maganin ruwa mai kama da juna.An yi amfani da lysate na tsawon minti 20 kuma an haxa shi tare da lysate da aka samu a mataki na baya.An tantance adadin furotin ta amfani da gwajin Micro BCA (Thermo Fisher Scientific) bisa ga umarnin masana'anta don hanyoyin microplate.An daskarar da samfuran da sauri cikin ruwa nitrogen kuma an adana su a -80 ° C.
Kusan 100 μg na furotin mai haɗin gwiwa mai narkewa an sarrafa shi ta amfani da ingantaccen tsarin shirye-shiryen tace tacewa (FASP) kamar yadda Wisniewski et al ya bayyana.69 A taƙaice, sunadaran suna haɗe tare da 200 µl na urea buffer (8 M urea a cikin 0.1 M Tris, pH 8.5), murƙushewa da rabi.An yi duk matakan centrifugation a 14,000 xg a 25 ° C.Rabin farko na lysate na gina jiki mai haɗin giciye an canza shi zuwa na'urar tacewa na 10 kDa Microcon centrifugal wanda aka sanye da membrane na Ultracel-10 (Merck), sannan ta hanyar centrifugation akan tacewa na mintuna 25.Sa'an nan kuma ƙara rabin na biyu na furotin zuwa tacewa kuma maimaita matakan guda ɗaya.An sake dawo da furotin ta ƙara 100 μl na 17 mM tris (2-carboxyethyl) phosphine hydrochloride (TCEP) a cikin buffer urea.An motsa farfadowa a kan thermomixer a 600 rpm na 30 min a 37 ° C.Bugu da ƙari, ginshiƙi ya kasance centrifuged kuma an rage raguwar furotin da aka haɗa ta hanyar amfani da 100 μl na 50 mM iodoacetamide a cikin buffer urea.An gudanar da maganin alkylation a dakin da zafin jiki na minti 20 a cikin duhu.Juya ginshiƙi, wanke bangon ginshiƙi sau 3 tare da buffer urea 100 µl, sa'an nan kuma a ɗaure.An yi wannan aikin sau 3 ta amfani da 100 μl na 100 mm ammonium bicarbonate.Kafin trypsinization, maye gurbin bututun tarin da sabo.Ƙara buffer narkewa mai ɗauke da 50 mM ammonium bicarbonate da 1 µl trypsin da aka diluted a cikin buffer trypsin (Promega).An kiyaye rabon trypsin da furotin a kusan 1:33, kuma an haɗa halayen narkewa cikin dare a 37 ° C. a cikin ɗaki mai ɗanɗano.An cire peptide mai haɗin gwiwa daga tacewa ta hanyar centrifugation na mintuna 25.An inganta farfadowar Peptide ta hanyar ƙara 50 μl na 0.5 M NaCl zuwa tacewa, sannan sai centrifugation na minti 25.
An yi amfani da ginshiƙan C18 Micro Spin (Harvard Apparatus) don lalata peptides masu haɗin gwiwar giciye da ke bin ka'idar da Bouchal et al.70 ya bayyana tare da ƙananan gyare-gyare.A taƙaice, an kunna ginshiƙan juyawa na C18 tare da wanke uku na 0.1% formic acid (FA) a cikin acetonitrile (AcN) (Merck) da wankin 0.1% FA.An shayar da ginshiƙi tare da 0.1% FA na mintuna 15.Load da samfurori cikin ginshiƙai kuma ku wanke sau 3 tare da 0.1% FA.Abubuwan peptides da aka lalata an binne su tare da matakin matakin mataki ta hanyar amfani da 50%, 80% da 100% AcN a cikin 0.1% FA.An bushe samfuran a cikin mai tattara bayanai na SpeedVac Plus (Eppendorf) har sai ragowar ruwan ya ɓace gaba ɗaya.An narkar da peptides na sama a cikin 100 μl na 0.08% trifluoroacetic acid a cikin 2.5% AcN kuma an auna abubuwan da aka tattara akan NanoDrop 2000 (Thermo Scientific).Kimanin 1 μg na peptide crosslinked kowane samfurin an yi allurar cikin tsarin LC-MS/MS.
An raba peptides masu haɗin giciye akan tsarin UltiMate 3000 RSLCnano LC (Thermo Scientific) wanda aka haɗa zuwa Orbitrap Exploris 480 mass spectrometer (Thermo Scientific).An tattara peptides masu haɗin kai akan ID na 300 µm, 5 mm tsayin µ-pre-column C18 ginshiƙin kamawa cike da C18 PepMap100 sorbent da 5 μm PepMap sorbent (Thermo Scientific).Load da famfon da aka saita a 5 µl/min 0.08% trifluoroacetic acid narkar da cikin 2.5% AcN.An raba peptides masu haɗin giciye akan ginshiƙin siliki mai haɗaɗɗiyar nazari tare da diamita na ciki na μm 75 da tsayin 150 mm, cike da 2 μm PepMap sorbent (Thermo Scientific).Matakan wayar hannu A da B sun ƙunshi 0.1% FA cikin ruwa da 0.1% FA a cikin acetonitrile, bi da bi.Ƙarfin yana farawa daga 2.5% B kuma yana ƙaruwa a layi zuwa 40% B fiye da mintuna 90, sannan zuwa 90% B a cikin mintuna 2 masu zuwa.An kiyaye tsarin tsarin wayar hannu a 90% B na mintuna 10 sannan ya ragu a layi-layi zuwa 2.5% B sama da mintuna 2.An daidaita ginshiƙi a 2.5% B don mintuna 8 kafin sake zagayowar gaba.peptides masu alaƙar giciye waɗanda aka ɓoye daga ginshiƙi na nazari an ionized a cikin tushen nanoelectrospray ionization (NSI) kuma an yi musu allura a cikin Exploris 480 mass spectrometer (Thermo Scientific).
Orbitrap Exploris 480 mass spectrometer yana aiki a cikin ingantaccen yanayin daidaita bayanai.An yi cikakken bincike a yanayin sashe a ƙuduri na 120,000 tare da saitunan kewayo daga m/z 350 Th zuwa m/z 2000 Th.An saita maƙasudin AGC na yau da kullun a 300% tare da matsakaicin lokacin shigarwa na 50ms.An kafa gano kololuwar monoisotopic don peptides.An saita siginar takurawa zuwa gaskiya idan an sami mafari kaɗan kaɗan.An saita ƙaramin ƙarfin ionic na mafari zuwa 5.0e3 kuma an haɗa jahohi na farko har zuwa +8 a cikin gwaje-gwajen.
Lokacin zagayowar tsakanin manyan sikanin a cikin yanayin daidaita bayanai an saita zuwa daƙiƙa 2.5.An saita keɓance taro mai ƙarfi zuwa 20 s bayan rarrabuwar farko na ion precursor.An saita taga keɓewar precursor zuwa 2th.Nau'in daidaitawar kuzarin karo tare da tsayayyen yanayin kuzarin karo an zaɓi a cikin binciken MS/MS da ya dogara da bayanai.Ƙarfin haɗari ya saita zuwa 30%.An saita ƙudurin Orbitrap zuwa 15,000 kuma AGC manufa zuwa 100%.Matsakaicin lokacin allura na al'ada an saita shi zuwa millise seconds 60.
Kafin bin diddigin hanyar sadarwar furotin-protein a cikin samfuran haɗin gwiwar giciye, mun sarrafa albarkatun albarkatun ta amfani da kunshin MaxQuant (Sigar 1.6.12.0) 26,27 don gano peptides / furotin da aka gano a cikin samfuran.Bugu da ƙari, an yi nazari na proteomic irin wannan akan samfuran Flo-1 da ba a haɗa su ba da aka bi da su ba tare da IFNα ba.An bincika bayanan MS/MS a cikin bayanan mutum na UniProt (www.uniprot.org) (wanda aka ɗora a ranar 12 ga Agusta, 2020, ya ƙunshi shigarwar 75,093) ta amfani da ingin binciken Andromeda27.An gudanar da binciken ba tare da nuna ƙayyadadden ƙwayar enzyme ba da kuma gyare-gyare daban-daban na deamidation (N, Q) da oxidation (M).An saita jurewar taro na farko a 20 ppm da ions samfur a 0.02 Da.An saita farkon da matsakaicin karkatar taro zuwa 10 ppm.Matsakaicin adadin peptide an saita shi a 4600 Da kuma an saita kamanceceniya tsakanin 7 da 25 amino acid (aa).An yi ƙarin nazarin ƙididdiga ta amfani da shirin Perseus (sigar 1.6.10.45).An ƙididdige abubuwan da ke cikin furotin ta hanyar daidaita girman girman furotin (ƙarfin LFQ; ƙididdigewa mara lakabi)27 kuma an canza ƙimar ƙimar zuwa Log2.An gina tarin sunadaran sunadaran da aka gano ta hanyar ƙarfin peptide su ta amfani da fakitin pheatmap (v1.0.12) a cikin R (v 4.1.2).An gudanar da nazarin haɓakar hanyar ta hanyar amfani da bayanan hanyar Reactome don sunadaran da aka yi wa IFNa waɗanda aka kunna fiye da sau huɗu idan aka kwatanta da samfuran da ba a kula da su ba.
Gano lysine (K) ko serine (S) takamaiman hanyoyin haɗin sinadarai na rukunin furotin da LC-MS/MS ke kula da su an yi su ta amfani da na'urar ganowa ta spectroscopic (SIM-XL) don peptides masu alaƙa (SIM-XL)29.Na farko, yiwuwar hulɗar tsakanin interferon-hade (IFN) DNA lalacewa juriya sa hannu (IRDS) genes an bincika ta amfani da bayanan furotin IRDS da aka bayyana a cikin Padariya et al.28.Nuna duk yanayi da maimaitawa na UniProt na ɗan adam gabaɗaya yana da ƙima sosai, don haka duk bayanan UniProt na ɗan adam (www.uniprot.org) (wanda aka sauke 12 ga Agusta 2020, ya ƙunshi shigarwar 75,093) akan maimaitawar IFNα.Daya daga cikin tacewa don babban amintaccen hulɗa.Wadannan ma'amala masu mahimmanci da aka samu an fadada su kuma an gwada su a duk maimaitawa da yanayi.
A cikin SIM-XL, an yi amfani da DSS don crosslinker (XL) kuma an saita nauyin nauyin XL da gyare-gyaren nauyi zuwa 138.06 da 156.07, bi da bi.Ana la'akari da wuraren amsawar haɗin kai masu zuwa: KK, KS da KN-TERM, ba tare da ions masu rahoto ba.Dukkanin precursor da guntu ppm an saita su zuwa 20 kuma an saita iyakar Xrea zuwa 0.15.An yi la'akari da Trypsin a matsayin ƙayyadaddun ƙayyadaddun bayanai, kuma an aiwatar da hanyar rarrabawar C-trap (HCD) mai ƙarfi.Ƙofar raguwar XCorr DB mai ƙarfi da ƙaramin adadin peptides don raguwar DB mai ƙarfi an saita zuwa 2.5 da 2, bi da bi.Sauran sigogi sune: yuwuwar monoisotope da yanke daidaituwar daidaituwa, mafi ƙarancin ragowar AA a kowane madauri da matsakaicin cajin igiya, da 3 maxima na rarrabuwa da aka rasa.An yi nazarin taswirorin 2D ɗin da aka dinka a cikin (SIM-XL) kuma an yi amfani da wakilcin hoto na xQuest28 don gina taswirorin 2D.Ana samar da hanyoyin haɗin furotin akan tsarin furotin a cikin PyMol (Tsarin zane-zane na PyMOL Molecular Graphics, sigar 2.0 Schrödinger, LLC).
An ƙirƙiri tsarin ƙirar furotin ta amfani da uwar garken Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2)11 ta amfani da ka'idodin ƙirar homology da aiwatar da "Hanya Hidden Markov".Phyre2 yana haifar da ƙirar ƙira bisa jeri jeri tare da sanannun sifofin sunadaran.Don H2BFS, HLA-A, HMGA1, LRCH4, da sunadaran MDN1, an yi amfani da tsarin samfuri 1kx552, 1kj349, 2eze55, 6hlu62, da 6i2665.Bugu da kari, an kuma yi la'akari da tsarin AlphaFold71 MX1, UBP18 da ROBO1.An hango tsarin furotin ta amfani da kunshin BIOVIA Discovery Studio Visualizer (Dassault Systèmes, BIOVIA, San Diego, CA, Amurka) da Kunshin Ayyukan Muhalli na Molecular (MOE; Chemical Computing Group Inc., Montreal, Quebec, Canada).